Prion diseases are among the most rapidly progressive neurodegenerative disorders and are characterized pathologically by extracellular prion aggregates, synaptic damage, neuronal loss, and severe astrogliosis in the brain and spinal cord. Prion aggregates spread through neuroanatomically connected brain regions, yet how prions physically spread from cell-to-cell is poorly understood. In vitro, prion aggregates form on the plasma membrane, in endosomes, and in multivesicular bodies, and are released in exosomes from chronically infected cells. A major goal of this application is to determine how intra-cellular vesicular prion trafficking contributes to inter-cellular prion spread through the central nervous system using in vitro and in vivo model systems. We have previously employed a broad range of approaches to track structurally diverse prions from axon terminals to neuronal cell bodies and have determined the biophysical properties of highly virulent prions that spread into the CNS. We discovered that small, subfibrillar and fibrillar prions were internalized by neurons through macropinocytosis. However, only the small, subfibrillar prions spread from extraneural sites into the brain. Thus, aggregate size underlies prion spread into the CNS. We also determined that post-translational modifications in the prion protein can alter aggregate packing arrangements and lead to the emergence of new prion strains. Finally, we found that autophagic clearance pathways were induced in muscle cells harboring prion aggregates. In this renewal, we aim to determine how the vesicular trafficking of prions in neurons and glia impacts prion spread through the CNS. In Specific Aim 1, we will define the physical properties of a prion that govern packaging into exosomes. In Specific Aim 2, we will identify key regulators of intracellular prion conversion and clearance in neurons and astrocytes by manipulating vesicular transit pathways. Additionally we will characterize vesicular regulatory protein expression in prion-infected humans and in mouse models. In Specific Aim 3, we will determine how cell-specific repression of early and late stages of vesicular trafficking modifies prion disease progression. These experiments are the first to probe the contribution of intra-vesicular prion trafficking pathways to prion spread in vivo, and will help unravel how vesicular transport impacts prion conversion, clearance, and rapid spread through the brain. The proposed studies are particularly important with the growing recognition of endosomal and lysosomal dysfunction occurring in Alzheimer?s and other neurodegenerative diseases, and with potential opportunities arising for therapeutic intervention in protein aggregate clearance pathways.